1. Field of the Invention
The present invention relates to methods for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide through three-dimensional interactions and immunoassay kits therefor.
2. Description of the Related Art
A multimerization of polypeptides constituting proteins has been generally known to be required for the function of proteins. However, the multimeric forms often cause diseases or disorders in some proteins. In particular, a protein exists as a monomer in normal conditions and is converted to a multimer (or aggregate form) in abnormal conditions {e.g., by the conversion to a misfolding form).
It has been well established that proteins that are misfolded and ultimately aggregated (or accumulated), i.e., that are not in their functionally relevant conformation are devoid of normal biological activity. The failure to fold correctly, or to remain correctly folded, gives rise to many different types of biological malfunctions and hence, to many different forms of diseases (Massimo Stefani, et al., J. Mol. Med. 81:678-699 (2003); and Radford S E, et al., Cell. 97:291-298 (1999)). Many diseases ultimately result from the presence in a living system of protein molecules with structures that are incorrect, i.e., that differ from those in normally functioning organisms.
For instance, the diseases or disorders associated with abnormal aggregation or misfolding of proteins include Alzheimer's disease, Creutzfeldt-Jakob disease, Spongiform encephalopathies, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis, Type II Diabetes, primary systemic amyloidosis, secondary systemic amyloidosis Fronto-temporal dementias, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and haemodialysis-related amyloidosis.
Early diagnosis of the aggregation-associated diseases has been intensively studied. However, there has not been suggested any process and approach to differentially detect multimeric (aggregating) forms from their monomeric (normal) forms.
Sporadic, variant, iatrogenic, and familial Creutzfeldt-Jakob diseases, kuru, Familial Fatal insomnia, and Gerstmann-Straussler-Scheinker syndrome in humans, scrapie in sheep and goats, feline spongiform encephalopathy in cat, mink spongiform encephalopathy, Chronic Wasting disease in deer, elk, and moose, and bovine spongiform encephalopathy in cattle are the fatal neurodegenerative diseases, due to transmissible spongiform encephalopathies (TSE) (Prusiner S. B. Proc. Natl. Acad. Sci. USA 95:13363-13383 (1998); and Hope J. Curr. Opin. Genet Dev. 10, 568-57 (2000)). Abnormal isoform or the scrapie form of prion protein (PrPSc) has been strongly suggested to the main culprit of TSE (Caughey B. Trends Biochem. Sci. 26:235-42 (2001)).
The normal form of the prion protein (PrPc), contains both an α-helical and a flexibly disordered portion and exists as a monomeric form (Zahn, R., et al., Proc. Natl. Acad. Sci. USA 97:145-150 (2000)), where the scrapie form (PrPSc) has highly β-sheet conformation and exists as a multimeric (aggregating) or at least dimer forms (Caughey, B., et al., J. Biol. Chem. 273:32230-35 (1998)). The conformational change from α-helical to β-sheet conformations is the central event of the disease that seems to be responsible for its neuropathology.
While PrPc is protease sensitive (PrPsen), PrPSc is partially resistant to proteolysis (PrPres) and prone to form high-molecular-weight aggregates (Bolton D. C. Lancet, 358:164-5 (2001)). This latter feature makes it difficult to analyze the conformational transition that leads to the formation of PrPres or to characterize it.
The method of protease K (PK) digestion has been used to discriminate the resistance of its various forms of PrP (scrapie form) by digesting the cellular form, leaving only the scrapie form to be detected in ELISA. However, the PK digestion method is being questioned. PrP conformation, concentration, tissue antibodies, digestion time and buffers could influence the PK sensitivity, which significantly reduces the reliability of the PK digestion method.
Therefore, there remains a need to develop a novel approach for differentially detecting multimeric form {e.g., PrPSc, scrapie form of PrP) from their monomeric forms {e.g., PrPc, cellular form of PrP) with much higher reliability and convenience.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.